The present invention relates to new lipid compounds and new compositions containing them. More particularly, the present invention relates to the use of said compounds or of said compositions to prepare a vector for transferring an active substance, in particular a therapeutically active substance comprising negative charges, in particular a polynucleotide, into a target cell, particularly a vertebrate cell, and more particularly a mammalian cell.
The transfer of a gene into a given cell is the very basis of gene therapy. This new technology, whose field of application is vast, makes it possible to envisage the treatment of serious diseases for which the conventional therapeutic alternatives are not very effective, or are even nonexistent, and applies to diseases which are either of genetic origin (hemophilia, cystic fibrosis, myopathy and the like) or acquired (cancer, AIDS and the like).
During the past 30 years, numerous tools have been developed which allow the introduction of various heterologous genes into cells, in particular mammalian cells. These different techniques may be divided into two categories. The first category relates to physical techniques such as microinjection, electroporation or particle bombardment which, although effective, are greatly limited to applications in vitro and whose implementation is cumbersome and delicate. The second category involves techniques relating to molecular and cell biology in which the gene to be transferred is combined with a vector of a biological or synthetic nature which promotes the introduction of said material.
Currently, the most effective vectors are viral, in particular adenoviral or retroviral, vectors. The techniques developed are based on the natural properties which these viruses have to cross the cell membranes, to escape degradation of their genetic material and to cause their genome to penetrate into the nucleus. These viruses have already been the subject of numerous studies and some of them are already used experimentally as vectors for genes in humans for the purpose, for example, of a vaccination, an immunotherapy or a therapy intended to make up for a genetic deficiency. However, this viral approach has many limitations, in particular because of the limited capacity for cloning into the viral genome, the risks of spreading in the host organism and in the environment the infectious viral particles produced, the risk of artefactual mutagenesis by insertion into the host cell in the case of retroviral vectors, and the high induction of immune and inflammatory responses in vivo during the therapeutic treatment, considerably limiting the number of administrations which can be envisaged (McCoy et al., 1995, Human Gene Therapy, 6, 1553-1560; Yang et al., 1996, Immunity, 1, 433-442). These numerous disadvantages, in particular in the context of a use in humans, have led several teams to develop alternative systems of transferring polynucleotides.
Several nonviral methods are currently available. By way of example, there may be mentioned coprecipitation with calcium phosphate, the use of receptors mimicking viral systems (for a review see Cotten and Wagner, 1993, Current Opinion in Bio-technology, 4, 705-710), or the use of polymers such as polyamidoamine (Haensler and Szoka, 1993, Bioconjugate Chem., 4, 372-379) or of polymer such as those presented in WO 95/24221 describing the use of dendritic polymers, the document WO 96/02655 describing the use of polyethyleneimine, or of polypropyleneimine and the documents U.S. Pat. No. 5,595,897 and FR 2,719,316 describing the use of conjugates of polylysine. Other non-viral techniques are based on the use of liposomes whose value as agent allowing the introduction, into cells, of certain biological macromolecules, such as for example DNA, RNA, proteins or certain pharmaceutically active substances, has been widely described in the literature. To this end, several teams have already proposed the use of cationic lipids which have a high affinity for cell membranes and/or nucleic acids. Indeed, although it has been shown, in the case of nucleic acids, that this type of macromolecule is capable of crossing the plasma membrane of some cells in vivo (WO 90/11092), it is nevertheless the case that the observed transfection efficiency is still highly limited, because of in particular the polyanionic nature of the nucleic acids which prevent their passage across the cell membrane, which itself has a negative net apparent charge. Since 1989 (Felgner et al., Nature, 337, 387-388), cationic lipids have been presented as molecules which are advantageous for promoting the introduction of large anionic molecules, such as nucleic acids, into certain cells. These cationic lipids are capable of complexing anionic molecules, thus tending to neutralize the negative charges on said molecules and to promote their coming close to the cells. Many teams have already developed various cationic lipids. By way of example, there may be mentioned DOTMA (Felgner et al., 1987, PNAS, 84, 7413-7417), DOGS or Transfectam(trademark) (Behr et al., 1989, PNAS, 86, 6982-6986), DMRIE and DORIE (Felgner et al., 1993, Methods 5, 67-75), DC-CHOL (Gao and Huang, 1991, BBRC, 179, 280-285), DOTAPxe2x96xa1 (McLachlan et al., 1995, Gene Therapy, 2,674-622) or Lipofectamine, as well as those described in Patent Applications WO9116024 or WO9514651
More particularly, Patent Application WO-A-9116024 describes cationic lipids of formula: 
in which:
R1 and R2 are in particular alkyl or alkenyl radicals;
Y1 and Y2 are radicals xe2x80x94OCH2xe2x80x94, xe2x80x94OC(xe2x95x90O)xe2x80x94 or xe2x80x94Oxe2x80x94;
R3 and R4 are alkyl or alkenyl radicals;
R5 is an alkylene chain;
R6 is C(xe2x95x90O)xe2x80x94(CH2)mxe2x80x94NHxe2x80x94, a diaminocarboxylic acid or xe2x80x94C(xe2x95x90O)xe2x80x94(CH2)mxe2x80x94NHxe2x80x94 bound to said diaminocarboxylic acid;
R7 is H, spermine, spermidine, histone, a protein, an amino acid or a polypeptide.
However, several studies (by way of examples, see Mahato et al., J. Pharm. Sci., 1995, 84, 1267-1271, Thierry et al., 1995, P.N.A.S., 92, 9742-9746) have demonstrated that the efficiency of transferring the anionic macromolecule into cells could vary depending in particular on the interaction between the complexes and the cell membranes, the cell considered, the lipid composition of the cationic compounds, the size of the complexes formed with the anionic molecules and more particularly the ratio between the positive and negative charges on the different components of said complex. The mechanisms which allow in particular the interaction of the complexes with the cell membranes and the transfer of the complexes into the cell are still to a large extent poorly understood and researchers proceed in their studies based on a highly empirical approach. Other factors such as, for example, the formation of the complexes, the stability, the behavior in vivo, or possibly their toxicity make, in addition, the choice of the lipids to priori non-obvious. It is consequently desirable to provide other cationic lipids possibly having improved properties or properties which are different from the cationic lipids already described.
The Applicant has now identified new lipid compounds, which can be provided in cationic form, useful in particular for transferring an active substance, in particular a therapeutically active substance, comprising negative charges, in particular a polynucleotide, into a target cell, whose use may be envisaged in particular in vivo in the context of a gene therapy.
Accordingly, the subject of the present invention is first of all a lipid compound of formula:
Rxe2x80x94HNxe2x80x94[xe2x80x94(CH2)mxe2x80x94NRxe2x80x94]nxe2x88x921xe2x80x94(CH2)mxe2x80x94NHxe2x80x94Rxe2x80x83xe2x80x83I
in which:
the R residues are, independently of each other, a hydrogen atom or a group of formula II: 
xe2x80x83in which:
R1 and R2 are, independently of each other, C6-C23 alkyl or alkenyl radicals, which are linear or branched, or radicals xe2x80x94C(xe2x95x90O)xe2x80x94(C6-C23) alkyl or xe2x80x94C(xe2x95x90O)xe2x80x94(C6-C23) alkenyl, which are linear or branched, aryl radicals, cycloalkyl radicals, fluoroalkyl radicals, polyethylene glycol groups, oxyethylene or oxymethylene groups which are optionally repeated, linear or branched, optionally substituted,
p is a positive integer from 1 to 4,
n is a positive integer from 1 to 6,
m is a positive integer from 1 to 6 which may be different for each motif xe2x80x94(CH2)m, and more particularly for each motif xe2x80x94(CH2)mxe2x80x94NRxe2x80x94 when n greater than 1,
the number of R groups of formula II being between 1 and 4.
The expression xe2x80x9calkenylxe2x80x9d is understood to mean that the carbon chain may comprise one or more double bond(s) along said chain.
According to a specific case, the invention relates to a compound chosen from the compounds of formulae:
H2Nxe2x80x94[xe2x80x94(CH2)mxe2x80x94NHxe2x80x94]nRxe2x80x83xe2x80x83III
RNHxe2x80x94[xe2x80x94(CH2)mxe2x80x94NH]nHxe2x80x83xe2x80x83IIIa
in which:
R is a group of formula II as defined above and
H2Nxe2x80x94[xe2x80x94(CH2)mxe2x80x94NR]nxe2x88x921xe2x80x94(CH2)mxe2x80x94NH2xe2x80x83xe2x80x83IIIb
xe2x80x83in which:
R has one of the meanings indicated for the formula I provided that at least one R is a group of formula II and for each of the formulae:
n is a positive integer from 1 to 6,
m is a positive integer from 1 to 6 which may be different for each motif xe2x80x94(CH2)m, and more particularly for each motif xe2x80x94(CH2)mxe2x80x94NRxe2x80x94 when n greater than 1.
According to a preferred case, the compounds of formula IIIb contain one or two R groups of formula II.
According to preferred embodiments of the invention, the variations presented below, taken in combination with each other or otherwise, will be chosen:
R1 and R2 are, independently of each other, linear xe2x80x94C(xe2x95x90O)-alkyl or linear xe2x80x94C(xe2x95x90O)-alkenyl radicals,
R1 and R2 are, independently of each other, xe2x80x94C(xe2x95x90O)-alkyl or xe2x80x94C(xe2x95x90O)-alkenyl radicals comprising from 12 to 20 carbon atoms, preferably 12, 16 or 18 carbon atoms, when the lipid comprises 1 or 2 R groups of formula II,
n is an integer chosen from the numbers 2, 3 or 4,
m is an integer chosen from the numbers 2, 3 or 4.
According to a specific case, it is possible to reduce the length of the alkyl or alkenyl chain so that R1 and R2 are radicals having 6 to 10 carbon atoms, but in this case compounds for which the number of R groups of formula II is equal to 2, 3 or 4 will be preferably chosen.
Preferably, the lipids according to the invention are chosen from the group consisting of the compounds of the following formulae: 
for which R1 and R2 are identical and are chosen from the stearoyl and oleoyl radicals.
The compounds according to the invention are prepared by reacting a compound of formula: 
in which:
R3 and R4 are protecting groups, in particular Fmoc (Grandas et al., 1989, Int. Journal pept. prot. Res. vol 33, 386-390), with an amine of formula:
R5NH[(xe2x80x94CH2)mxe2x80x94NR5]nxe2x88x921xe2x80x94(CH2)mNHR5xe2x80x83xe2x80x83VI
m and n having the same meaning as for the formula I,
R5 being a protecting group, in particular t-butoxycarbonyl (BOC) or a hydrogen atom, at least one of the R5 radicals and at most four of the R5 radicals corresponding to the hydrogen atom.
The functional groups Nxe2x80x94R3 and xe2x80x94Nxe2x80x94R4 are then deprotected so as to bind, by amidation or alkylation, the radicals R1 and R2 in a known manner, in particular by the action of the corresponding xe2x80x94N-hydroxy-succinimide ester.
The compound obtained is deprotected in the presence of trifluoroacetic acid.
The amines of formula VI are prepared in a known manner.
In the case where the compound of formula VI is 1-4-di-boc-spermidine, reference will be made to the examples indicated below in order to know the practical modalities for the synthesis. The processes described are applicable in general to the syntheses of the compounds according to the invention subject to adaptations within the capability of persons skilled in the art.
However, the compounds of the invention cannot be limited to those obtained by the modes of preparation described above.
The compounds according to the invention may, in addition, be substituted. Such substitutions may in particular consist of a labeling molecule (see labeling molecules in U.S. Pat. No. 4,711,955) which makes it possible, for example, to visualize the distribution of the compounds or of the complexes containing them after administration in vitro or in vivo, a cell targeting molecule or an anchoring molecule. The invention consequently also relates to a compound as presented above, conjugated with one or more targeting components, also called ligands of interest, via the intermediacy of at least a) one of the carbon atoms, in particular chosen from those present on the groups R1 and/or R2, or b) one of the secondary or primary nitrogen atoms of the polyamine chain or of the diaminocarboxylic acid. Such components may allow targeting to a specific cell type, facilitate penetration into the cell, lysis of the endosomes or alternatively intracellular transport and are widely described in the literature. They may be, for example, all or part of sugars, peptides (GRP peptide, Gastrin Releasing Peptide, for example), oligonucleotides, lipids, hormones, vitamins, antigens, antibodies, ligands specific for membrane receptors, ligands capable of reacting with an anti-ligand, fusogenic peptides, nuclear localization peptides, or a combination of such compounds. There may be mentioned more particularly the galactosyl residues which make it possible to target the asyaloglycoprotein receptor at the surface of hepatic cells, the fusogenic peptide INF-7 derived from the influenza virus hemagglutinin subunit HA-2 (Plank et al., 1994, J. Biol. Chem. 269, 12918-12924) or a nuclear localization signal derived from the SV40 virus T antigen (Lanford and Butel, 1984, Cell 37, 801-813) or the Epstein Barr virus EBN-1 protein (Ambinder et al., 1991, J. Virol. 65, 1466-1478).
Such conjugates can be easily obtained by techniques widely described in the literature, and more particularly by chemical coupling, in particular using protecting groups such as trifluoroacetyl or Fmoc or Boc, onto the polyamine and more particularly using one or more orthogonal protecting groups such as those described in Protective Groups in Organic Synthesis (p. 309-406, 1991, eds. T. W. Greene, P. G. M. Wuts, Wiley) onto the polyamine or the diaminocarboxylic acid. The selective deprotection of a protecting group then makes it possible to couple the targeting component, and the lipid is then deprotected. It should be stated, however, that the substitution of the nonreactive groups such as the carbon atoms in the CH or CH2 groups will be carried out during synthesis of the compounds of the invention by methods known to a person skilled in the art, whereas the reactive groups, such as the primary or secondary amines, may be the subject of substitutions on the neosynthesized lipids of the invention.
According to an advantageous case of the invention, said compound is in a cationic form, that is to say that it is in a form which is protonated by binding of a proton onto one or more nitrogen atoms present on the polyamine chain. In this case, said cationic lipid is combined with one or more biologically acceptable anions, such as for example the trifluoroacetate, halide, monomethylsulfate, acetate or phosphate, iodide, chloride, or bromide anion and the like. It is also possible to obtain compounds in cationic form by substitution of the amines, for example, with a methyl or ethyl radical, and the like.
According to another aspect, the invention also relates to a composition comprising at least one compound as described above and optionally at least one adjuvant capable of enhancing the formation of the complex between a said compound and an active substance, or of enhancing the function of these complexes toward the cell.
Preferably, such an adjuvant will be a neutral or zwitterionic lipid, such as for example a lipid which is or is derived from a triglyceride, a diglyceride, cholesterol (see for example U.S. Pat. No. 5,438,044), in particular, a neutral or zwitterionic lipid which is or is derived from a phosphatidylethanolamine (PE), phosphatidylcholine, phosphocholine, sphyngomyelin, ceramide or cerebroside. Advantageously, dioleoylphosphatidylethanolamine (DOPE) will be chosen.
The weight ratio between the compound of the invention and the neutral or zwitterionic lipid is generally between 0.1 and 10, it being understood that this ratio may vary depending on the nature of the components considered. Persons skilled in the art have sufficient knowledge to allow these minor adaptations. It is also possible to use a mixture of neutral and/or zwitterionic lipids or alternatively a mixture of cationic lipids and neutral and/or zwitterionic lipids.
The invention relates, in addition, to a complex comprising at least one compound or at least one composition as described above and at least one active substance, in particular a therapeutically active substance, comprising at least one negative charge. According to a variant of the invention, said complex may, in addition, contain one or more cationic amphiphilic agents such as those described in the literature of which examples were provided above.
According to a specific embodiment, said active substance is chosen from nucleic acids and proteins. Preferably, the active substance of the complex according to the invention is a polynucleotide, said compound or said composition then making it possible to enhance the transfecting power of the polynucleotide in a cell.
xe2x80x9cPolynucleotidexe2x80x9d is understood to designate a DNA and/or RNA fragment which is double-stranded or single-stranded, linear or circular, natural, isolated or synthetic, designating a precise succession of nucleotides, which are modified or otherwise (see by way of example U.S. Pat No. 5,525,711), labeled or otherwise (see for example U.S. Pat. No. 4,711,955 or EP 302175), making it possible to define a fragment or a region of a nucleic acid without size limitation. Polynucleotide is understood to designate in particular a cDNA, a genomic DNA, a plasmid DNA, a messenger RNA, an antisense RNA, a ribozyme, a transfer RNA, a ribosomal RNA or a DNA encoding such RNAs. xe2x80x9cPolynucleotidexe2x80x9d or xe2x80x9cnucleic acidxe2x80x9d are synonymous terms in the context of the present application. xe2x80x9cAntisensexe2x80x9d is understood to designate a nucleic acid having a sequence complementary to a target sequence, for example an mRNA sequence for which it is sought to block the expression by hybridization with the target sequence; xe2x80x9csensexe2x80x9d is understood to designate a nucleic acid having a sequence homologous or identical to a target sequence, for example a sequence which binds to a proteinaceous transcription factor and which is involved in the expression of a given gene.
According to a specific embodiment of the invention, said polynucleotide comprises a gene of interest and components allowing the expression of said gene of interest. In this embodiment, said polynucleotide is advantageously in the form of a plasmid. The components allowing expression are all the components allowing the transcription of said DNA fragment into RNA (antisense RNA or mRNA) and the translation of the mRNA into a polypeptide. They are in particular promoter sequences and/or regulatory sequences which are effective in said cell, and optionally the sequences required to allow excretion or expression of said polypeptide at the surface of the target cells. By way of example, there may be mentioned promoters such as the promoters of the viruses RSV, MPSV, SV40, CMV or 7.5k, of the vaccinia virus, the promoters of the gene encoding muscle creatine kinase, actin, or pulmonary surfactant. It is, in addition, possible to choose a promoter sequence specific for a given cell type or which can be activated under defined conditions. The literature provides a large amount of information relating to such promoter sequences. Moreover, said polynucleotide may comprise at least two sequences, which are identical or different, exhibiting a transcriptional promoter activity and/or at least two coding DNA sequences, which are identical or different, situated, relative to each other, contiguously, far apart, in the same direction or in the opposite direction, as long as the transcriptional promoter function or the transcription of said sequences is not affected. Likewise, it is possible to introduce into this type of nucleic acid construct xe2x80x9cneutralxe2x80x9d nucleic sequences or introns which do not affect transcription and are spliced before the translation step. Such sequences and their uses are described in the literature. Said polynucleotide may also contain sequences required for intracellular transport, for replication and/or for integration. Such sequences are well known to persons skilled in the art. Moreover, the polynucleotides according to the present invention may also be polynucleotides which are modified such that it is not possible for them to become integrated into the genome of the target cell or polynucleotides which are stabilized with the aid of agents such as, for example, spermine.
In the context of the present invention, the polynucleotide may be homologous or heterologous to the target cell. It may be advantageous to use a polynucleotide which encodes all or part of a polypeptide, in particular a polypeptide having a therapeutic or prophylactic activity, and more particularly an immunogenic activity of the cellular or humoral type. The term polypeptide is understood without restriction as to its size or its degree of modification (for example glycosylation). There may be mentioned, by way of examples, the genes encoding an enzyme, a hormone, a cytokine, a membrane receptor, a structural polypeptide, a polypeptide forming a membrane channel, a transport polypeptide, an adhesion molecule, a ligand, a factor for regulation of transcription, of translation, of replication, or of the stabilization of the transcripts, or an antibody, such as for example the gene encoding the CFTR protein, dystrophin, factor VIII or IX, E6/E7 of HPV, MUC1, BRAC1, xcex2-interferon, xcex3-interferon, interleukin (IL)2, IL-4, IL-6, IL-7, IL-12, tumor necrosis factor (TNF) type alpha, GM-CSF (Granulocyte Macrophage Colony Stimulating Factor), the Herpes Simplex virus type 1 (HSV-1) tk gene, the gene associated with retino-blastoma or p53 or all or part of immunoglobulins, such as the fragments F(ab)2, Fabxe2x80x2, Fab or the anti-idiotypes (U.S. Pat. No. 4,699,880). This list is of course not limiting and other genes may be used.
According to a preferred embodiment, the complexes according to the invention are small in size (less than 500 nm, advantageously less than 200 nm and preferably less than 100 nm).
Moreover, the transfection experiments carried out show that advantageously the weight ratio of the lipid compound according to the invention to said polynucleotide is 0.01 to 100. The optimum ratio is between 0.05 and 10.
The invention also relates to a process for preparing the complexes of cationic compounds/anionic active substances, said process being characterized in that one or more lipids in cationic form or a composition according to the invention whose lipid is in cationic form are brought into contact with one or more active substances comprising at least one negative charge and in that said complex is recovered, optionally after a purification step. It also relates to the kits for preparing such complexes comprising one or more lipids or one or more compositions according to the invention.
In a first instance, according to a first variant, one or more cationic compounds are dissolved with an appropriate quantity of solvent or mixture of solvents which are miscible in water, in particular ethanol, dimethylsulfoxide (DMSO), or preferably a 1:1 (v:v) ethanol/DMSO mixture, so as to form lipid aggregates according to a known method described, for example, in Patent Application WO-A-9603977, or according to a second variant, are suspended with an appropriate quantity of a solution of detergent such as an octylglucoside such as n-octyl-xcex2-D-glucopyranoside, or 6-O-(N-heptylcarbomoyl)-methyl-xcex1-D-glucopyranoside.
The suspension may then be placed in a buffer medium and mixed with a solution of active substance comprising negative charges.
In the case where it is desirable that a neutral or zwitterionic lipid is present in the final complex, a film is formed, in the known manner, prior to the dissolution in the solvent which is miscible with water or in the solution of detergent, with a mixture containing a said cationic compound and a said neutral or zwitterionic lipid, such as for example DOPE.
One of the important characteristics of the process consists in the choice of the ratio between the positive charges of the cationic lipid and the negative charges of the active substance.
Without wishing to be limited by a specific ratio, quantities of the different charges will be chosen so that the ratio between the number of positive charges of the compound or of the cationic composition and the number of negative charges of the active substance is between 0.05 and 20, in particular between 0.1 and 15, and preferably between 5 and 10.
This ratio between the number of positive charges of the cationic compound(s) and/or composition(s) and the number of negative charges of said active substance also constitutes an advantageous characteristic of the complex according to the invention.
The calculation to arrive at such a ratio will take into consideration the negative charges carried by the active substance and the quantity of compound necessary to satisfy the ratio indicated above will be adjusted. The quantities and the concentrations for the other components are adjusted according to their respective molar masses and the number of their positive and/or negative charges.
In the case of the second variant, subsequent dialysis may optionally be carried out in order to reduce the detergent and to recover the complexes. The principle of such a method is for example described by Hofland et al. (1996, PNAS 93, p 7305-7309) and in chapter II of the Philippot et al. document (G. Gregoriadis, 81-89, CRC Press 1993).
It has been shown that the first variant leads to excellent results in terms of the size of the complexes obtained.
According to a third variant, one or more cationic compositions or compounds are suspended in a buffer and then the suspension is subjected to sonication until visual homogeneity is obtained. The lipid suspension is then extruded through two microporous membranes under appropriate pressure. The lipid suspension is then mixed with a solution of active substance comprising negative charges. In the case where a neutral lipid is present in the complex, a film of the mixture of cationic lipid and neutral lipid such as DOPE is formed in a known manner prior to the preparation as a suspension. The same remarks relating to the ratio between the positive charges of the cationic lipid and the negative charges of the active substance as those indicated in the first variant are applicable to this third variant. This so-called sonication-extrusion technique is well known in the art.
The characteristics of the complexes formed may be evaluated by several means which make it possible to determine, for example:
the state of complex formation with the active substance, in particular by identification of the free nucleic acids by agarose gel electrophoresis in the case where the substances are nucleic acids,
the size of the particles by a quasi-elastic scattering of light,
the absence of precipitation over the long term.
The object of the present invention is also the complexes obtained using the processes listed above.
The invention also relates to the use of a compound, of a composition or of a complex according to the invention to transfer at least one active substance, especially a therapeutically active substance, more particularly a nucleic acid, into target cells, in vitro, ex vivo or in vivo, more particularly in vivo.
xe2x80x9cTarget cellsxe2x80x9d according to the invention is understood to mean prokaryotic cells, yeast cells and eukaryotic cells, plant cells, human or animal cells, and in particular mammalian cells. Cancer cells should, moreover, be mentioned. In vivo, the invention may be applied at the level of the interstitial or luminal space of tissues such as the lungs, trachea, skin, muscle, brain, liver, heart, spleen, bone marrow, thymus, bladder, lymph, blood, pancreas, stomach, kidney, ovaries, testicles, rectum, peripheral or central nervous system, eyes, lymphoid organs, cartilages and endothelium. According to an advantageous choice of the invention, the target cell will be a muscle cell, a hematopoietic stem cell or alternatively a cell of the airways, more particularly a tracheal or pulmonary cell, and advantageously a cell of the respiratory epithelium.
The complexes according to the invention can be used as a medicament for curative, preventive or vaccinal purposes. Accordingly, the subject of the invention is also the complexes of the invention as a medicament for curative, preventive or vaccinal purposes. Such complexes may be used in a method of therapeutic treatment which consists in transferring at least one therapeutically active substance, in particular a polynucleotide, into target cells, in particular a mammalian cell, and more precisely a muscle cell, a hematopoietic stem cell, or a cell of the airways, more particularly a tracheal or pulmonary cell, or a cell of the respiratory epithelium.
A compound according to the invention is most particularly advantageous for transferring a nucleic acid into a muscle cell or a pulmonary cell. It is the compound noted pcTG37 (see example).
More widely, the present invention also relates to a process for introducing an active substance comprising negative charges into a cell in particular in vitro, characterized in that cells, in particular cultured on an appropriate medium are brought into contact with a complex cationic compound/active substance comprising at least one negative charge according to the invention, in particular in the form of a suspension of complexes. After a certain incubation time, the cells are washed and recovered. The introduction of the active substance may be checked (optionally after lysis of the cell) by any appropriate means.
The process of introduction is well known per se. The term xe2x80x9cintroductionxe2x80x9d is understood to mean that the active substance comprising negative charges is transferred into the cell and is located, at the end of the process, inside said cell or at the level of the membrane thereof. In the case where the active substance is a nucleic acid, reference will be made more particularly to xe2x80x9ctransfectionxe2x80x9d. In this case, the verification of the transfection of the nucleic acid can be carried out by any appropriate means, for example by measuring the expression of the gene considered or the concentration of the expressed protein.
The invention relates more particularly to the use of a compound, of a composition or of a complex according to the invention for the preparation of a medicament for curative, preventive or vaccinal purposes, intended for the treatment of the human or animal body, in particular by gene therapy.
According to a first possibility, the medicament may be administered directly in vivo (for example into a muscle, into the lungs by aerosol and the like). It is also possible to adopt the ex vivo approach which consists in collecting cells from the patient (bone marrow stem cells, peripheral blood lymphocytes, muscle cells and the like), transfecting them in vitro according to the present invention and readministering them to the patient.
The complexes according to the invention may be administered by the intramuscular, intratracheal, intranasal, intracerebral, intrapleural, intratumoral, intracardiac, intragastric, intraperitoneal, epidermal, intravenous or intraarterial route by a syringe or by any other equivalent means, systems suitable for the treatment of the airways or of the mucous membranes such as inhalation, instillation or aerosolization. There may also be mentioned the modes of administration by application of a cream, by oral administration or any other means known to the person skilled in the art and applicable to the present invention.
It is also within the scope of the invention to target specific organs or tissues by administration, in particular by the intravenous route, of a complex according to the invention prepared so as to adjust the ratio of the compound or to the composition/therapeutically active substance in said complex, the apparent charge of the complex (see in particular Liu et al., 1997, Gene Therapy, 4, 517-523; Thierry et al., 1995, P.N.A.S., 92, 9742-9746).
The invention also relates to a method of gene therapy consisting in administering to a patient an appropriate quantity of a composition according to the invention. According to the present invention and in the context of gene therapy in vivo, it is possible to repeat several times, in a given patient, the method as proposed without any major immune reaction being elicited against one of the compounds administered. The administration may take place in a single dose or repeated once or several times after a certain time interval. The repeated administration would make it possible to reduce the quantity of therapeutically active substance, more particularly of DNA, to be administered for a given dose. The appropriate route of administration and dosage vary according to various parameters, for example the individual or disease to be treated or alternatively the polynucleotide to be transferred.
The invention relates more particularly to a pharmaceutical preparation comprising at least one complex as described above, optionally containing, in addition, at least one adjuvant capable of stabilizing said pharmaceutical preparation for the purpose of its storage for example and/or of enhancing the transfecting power of said complex. Such an adjuvant could, for example, be chosen from the group consisting of chloroquine, a protic polar compound chosen in particular from propylene glycol, polyethylene glycol, glycerol, ethanol, 1-methyl-L-2-pyrrolidone or derivatives thereof, or an aprotic polar compound chosen in particular from dimethyl sulfoxide (DMSO), diethyl sulfoxide, di-n-propyl sulfoxide, dimethyl sulfone, sulfolane, dimethylformamide, dimethylacetamide, tetramethylurea, acetonitrile or derivatives thereof. Likewise, said preparation may contain a pharmaceutically acceptable carrier allowing its administration to humans or animals.
In the context of the use of a method of treatment in vivo according to the present invention, it is, in addition, possible to carry out, before the administration of a pharmaceutical preparation as described above, a treatment of the patient designed to observe a temporary depletion of the macrophages making it possible to enhance the transfection rate. Such a technique is described in the literature; see in particular Van Rooijen et al., 1997, TibTech, 15, 178-184.
Finally, the invention relates to a cell transfected with a complex as defined above, particularly a prokaryotic cell, a yeast cell or eukaryotic cell, especially an animal cell, in particular a mammalian cell, and more particularly a cancer cell. According to a preferred case of the invention, said cell is a cell of the airways, more particularly a tracheal or pulmonary cell, and advantageously a cell of the respiratory epithelium.
the examples below illustrate the invention without limiting it in any manner.